This project aims to understand the structural mechanisms underlying high-speed chromosomal replication by replicative DMA polymerases, which consist of a catalytic core, a sliding clamp protein (beta clamp in bacteria, PCNA in eukaryotes) that tethers the core to DMA, and a heteropentameric ATP- dependent clamp loader complex (gamma complex in bacteria, RFC in eukaryotes) that loads the clamps onto DNA and coordinates the efficient replication of both the leading and the lagging strands. This project has previously established the three-dimensional structures of sliding clamps and clamp loaders, and now seeks to extend our understanding of clamp loader mechanism by determining crystal structures of clamp loaders bound to ATP analogs and DNA. The crystal structures of ATP-activated clamp loaders bound to open forms of the sliding clamps will also be determined. These studies will focus on clamp loaders and clamps from E. coli, the thermophilic eubacterium Aquifex aeolicus, the archaeon Methanococcus janschii and the eukaryote S. cerevisiae. High yield bacterial expression systems have been developed for all of these protein complexes. The project also includes structural characterization of the catalytic subunits of the replicative polymerases. Building on the novel structure of the catalytic (alpha) subunit of E. coli DNA polymerase III, recently determined as part of this project, the structure of a DNA complex of the alpha- subunit and the beta clamp will be pursued. Expression systems for eurkaryotic DNA polymerases will be optimized and these proteins prepared for crystallization. The mechanism by which the C-terminal domain of one subunit of the bacterial clamp loader, known as tau-C, promotes complete Okazaki fragment synthesis and efficient polymerase switching from one Okazaki fragment to the next one will be investigated by determining the structure of tau-C alone and in complex with the alpha subunit and DNA. Single molecule experiments using fluorescently labeled clamp-loader complexes and fluorescent nucleotide analogs will be carried out to define the mechanism by which ATP binding promotes the opening of the clamp, and how DNA promotes ATP hydrolysis and closure of the clamp. The structural and mechanistic information resulting from this work will facilitate the development of specific inhibitors of replication, either as antibiotics for the replicases from pathogenic bacteria, or as drugs that block human cell division in cancer.